Despite the success of combination antiretroviral therapy (cART) in children, HIV is remarkably persistent, except in one well-documented case, in which very early therapy likely resulted in viral eradication. HIV-1 persistence during cART is due to infection of long-living populations of CD4+ T cells. Early initiation of cART has been associated with lower numbers of infected cells and with restricted viral diversity in adults and older children [1, 3], but little is known about HIV-1 populations genetics in children who initiatd cART during infancy. It is possible that infants who initiated cART when the viral population was largely homogeneous have very limited viral diversity and no evidence of viral evolution over time. Better characterization of HIV-1 population genetics in infants initiated on cART at differen times after infection is needed to optimize HIV curative strategies. Recent evidence suggests that some HIV-1 infected CD4+ cells may have a proliferation and survival advantage when HIV integrations are in host genes related to growth regulation. Whether such clonal expansions emerge in children initiated on early cART is unknown. To fill these critical knowledge gaps, we propose the investigation of HIV diversity, using single genome sequencing (SGS) as well as viral integration sites, using a novel integration sites assay (ISA), in longitudinal samples (from baseline to 7-11 years post therapy initiation) in 30 children form a cohort of early antiretrovira treated HIV-1 infected children from the Children with HIV Early treatment (CHER) study. In patients who have sufficient HIV-1 diversity we will also link viral sequences integrated in the CD4+ cells with virus circulating in the blood plasma during and after treatment to reveal the specific proviral sources of persistent and/or rebound viremia. This has not been accomplished before. We expect to find a few children with very limited viral diversity and who have an increase in identical viral sequences (emanating from a few proliferating CD4+ cells populations, each with an identical integration site). In these patients, we will use a novel digitl droplet PCR (ddPCR) assay of specific integration sites with primers directed across the host-proviral junction to study the expansion and decline of CD4+ cells having a specific integrated provirus. Such results will provide critical information on the size, dynamics, and persistence of the HIV-1 reservoir. This study will be conducted as collaboration between Stellenbosch University, South Africa and the National Cancer Institute, U.S. The SGS, ISA and ddPCR technologies and bioinformatic algorithms to analyse the ISA data, developed at the NCI, will be transferred to South Africa, where the majority of the SGS and ISA assays will be performed. This technology transfer will add important research capabilities in S.A. that currently do not exist. This collaboration will better inform strategies aimed at controlling or curing HIV infectio without continued cART, such as therapeutic vaccination with HIV-1 antigens that adequately represent persistent HIV-1 populations.